Accumulation of reactive oxygen species (ROS) by hyperglycemia is a main cause of clinical complications during diabetes and obesity. Mitochondria are known to be a major source of ROS generation in hyperglycemic conditions. While perturbation of mitochondrial electron transport system (ETS) increases ROS generation, the causes of ETS deregulation are not fully understood. We observed mitochondrial fragmentation when cells were incubated with high glucose, which is mediated by mitochondrial fission proteins. Mitochondrial fission is essential for proper mitochondrial function, as deregulation of this process results in cell death. Conversely, inhibition of mitochondrial fission prevents apoptotic progression. The mitochondrial outer membrane protein hFis1 recruits the membrane severing protein DLP1 to mediate mitochondrial fission. We found that the tetratricopeptide repeat (TPR) motif of hFis1 binds to DLP1. In this application, we will test the novel concept that extensive fragmentation of mitochondria by high glucose concentrations alters membrane dynamics and proper structural organization of ETS, leading to increased ROS generation. Therefore, the Central Hypothesis of this application is that the mitochondrial fission machinery is an integral part of hyperglycemia-induced ROS generation under diabetic conditions and a potential target for preventing ROS-mediated cytotoxicity. We propose two Specific Aims that are focused on defining and developing a novel target and strategy to prevent mitochondrial ROS toxicity in diabetic hyperglycemia. First, we will define mitochondrial fission machinery as a modulating factor for mitochondrial ROS generation. We will examine the effects of increased and decreased mitochondrial fission on ROS generation by manipulating mitochondrial fission factors. Second, we will develop inhibitory factors for mitochondrial fission to reduce mitochondrial deformation and ROS toxicity. We will define the region of DLP1 that binds to the TPR region of hFis1. The short peptide binding to the hFis1 TPR is expected to block mitochondrial fission thus ROS toxicity. This study will provide insight into how changes in mitochondrial morphology participate in regulating ROS production and contribute to developing a yet unrecognized, novel target for preventing hyperglycemia-induced mitochondrial ROS toxicity in diabetes and obesity.